CN112779606A - Method for On-DNA Mannich reaction - Google Patents
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Abstract
本发明涉及一种On‑DNA曼尼希反应的方法，该方法以On‑DNA醛基化合物为底物，与活泼氢化合物和胺基化合物反应得到On‑DNA产物。该方法能够在有机溶剂/水相的混合溶剂中进行，环境友好；且能够大规模的引入醛、胺和含有活泼氢的化合物作为合成模块，适合使用多孔板的DNA编码化合物的合成。The invention relates to a method for On-DNA Mannich reaction, which takes On-DNA aldehyde-based compound as a substrate, reacts with active hydrogen compound and amine-based compound to obtain On-DNA product. The method can be carried out in a mixed solvent of organic solvent/aqueous phase, which is environmentally friendly; and can introduce aldehydes, amines and compounds containing active hydrogen as synthesis modules on a large scale, and is suitable for the synthesis of DNA-encoded compounds using multi-well plates.
Description
Technical Field
The invention belongs to the technical field of coding compound libraries, and particularly relates to a method for constructing an On-DNA beta-aminocarbonyl compound through an On-DNA Mannich reaction.
Background
In drug development, especially new drug development, high-throughput screening for biological targets is one of the main means for rapidly obtaining lead compounds. However, traditional high throughput screening based on single molecules requires long time, large equipment investment, limited number of library compounds (millions), and the building of compound libraries requires decades of accumulation, limiting the efficiency and possibility of discovery of lead compounds. The recent DNA-encoded compound library technologies (WO2005058479, WO2018166532, CN103882532) combine the technologies of combinatorial chemistry and molecular biology, add a DNA tag to each compound on the molecular level, and synthesize up to hundred million levels of compound libraries in a very short time, which is a trend of the next generation compound library screening technology, and begin to be widely applied in the pharmaceutical industry, resulting in many positive effects (Accounts of Chemical Research,2014,47, 1247-.
The DNA coding compound library can rapidly generate a giant compound library through combinatorial chemistry, and can screen out a lead compound with high flux, so that the screening of the lead compound becomes unprecedented rapidness and high efficiency. One of the challenges in constructing libraries of DNA-encoding compounds is the need to synthesize chemically diverse small molecules on DNA in high yields. Because DNA can be kept stable under certain conditions (solvent, pH, temperature and ion concentration), the On-DNA reaction applied to the construction of the DNA coding compound library also needs higher yield. Therefore, the reagent type, reaction type and reaction condition of the chemical reaction (On-DNA reaction for short) carried out On DNA directly influence the richness and selectivity of the DNA coding compound library. Therefore, the development of chemical reactions compatible with DNA is also a long-term research and research direction of the current DNA coding compound library technology, and the application and commercial value of the DNA coding compound library are directly influenced.
Beta-amino-carbonyl compounds are widely found in natural products and drug molecules, and thus the introduction of beta-amino-carbonyl compounds into a library of DNA-encoding compounds is of great importance. At present, no method for constructing the On-DNA beta-aminocarbonyl compound by the On-DNA aldehyde compound exists, so that the development of aqueous phase Mannich reaction of the On-DNA is needed to efficiently and quickly construct the beta-aminocarbonyl compound.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for constructing a beta-aminocarbonyl compound by an On-DNA Mannich reaction, which has the advantages of mild reaction conditions and simple post-treatment, is suitable for the production of a DNA coding compound library, and can obviously improve the molecular diversity of the compound library.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
an On-DNA Mannich reaction method, the reaction takes On-DNA aldehyde compound as raw materials, react with active hydrogen compound and amidogen compound to get On-DNA product; the structural formula of the On-DNA aldehyde compound is DNA-R1-CHO, the structural formula of the amino compound is R2R2 ’NH2The structural formula of the active hydrogen compound is
The structural formula of the On-DNA product is shown in the specification
Wherein the DNA in the structural formula comprises a single-stranded or double-stranded nucleotide chain obtained by polymerizing artificially modified and/or unmodified nucleotide monomers, and the nucleotide chain is connected with R through one or more chemical bonds or groups1Connecting; the length of the DNA is 10-200 bp.
Wherein, the DNA and R in the structural formula1Linked by a chemical bond or multiple chemical bonds or groups; when a chemical bond is present, it means DNA and R in the structural formula1Directly connecting; when there are more than one chemical bond or group, it means a junctionDNA and R in the formula1Are connected with a plurality of chemical bonds at intervals, for example, DNA and R1Through a methylene group (-CH)2-) are linked, i.e. linked by two chemical bonds; or DNA and R1The amino group of the DNA is connected with the amino group of the DNA through a carbonyl (-CO-) and is also connected through two chemical bonds; or DNA and R1Through a methylene carbonyl group (-CH)2CO-) is attached to the amino group of the DNA, i.e., by three consecutive chemical bonds.
Said R1Is a group with the molecular weight below 1000 directly connected with DNA and aldehyde group carbon atom or no group;
said R2、R2' is hydrogen or a group having a molecular weight of 1000 or less directly bonded to the nitrogen atom of the amino group;
said R3、R3' is hydrogen or a group having a molecular weight of 1000 or less directly bonded to the carbon atom on which the active hydrogen is present;
the EWG is an electron-withdrawing group with the molecular weight of below 1000 and directly connected with a carbon atom.
Preferably, the method comprises the following steps: said R1、R2、R2＇、R3、R3Each independently selected from an alkyl group, a substituted alkyl group, a 5-to 10-membered aryl group, a substituted 5-to 10-membered aryl group, a 5-to 10-membered aromatic heterocyclic group, and a substituted 5-to 10-membered aromatic heterocyclic group; wherein the alkyl is C1～C20Alkyl or C3～C8A cycloalkyl group; the number of substituents of the substituted alkyl group is one or more; the substituent of the substituted alkyl is one or more independently selected from halogen, carboxyl, nitro, alkoxy, halogenated phenyl, phenyl and alkyl phenyl; the number of the substituent for substituting the 5-to 10-membered aryl is one or more, and the substituents for substituting the 5-to 10-membered aryl are independently selected from halogen, cyano, nitro, carboxyl, alkoxy and C1～C20One or more of alkyl and trifluoromethyl; the number of the substituent(s) for substituting the 5-to 10-membered aromatic heterocyclic group is one or more, and the substituent(s) for substituting the 5-to 10-membered aromatic heterocyclic group are independently selected from the group consisting of halogen, cyano, nitro, carboxyl, alkoxy, C1～C20One or more of alkyl and trifluoromethylSeed growing;
said EWG is-NO2、-C(O)R、-CN、-C(O)OR、R-S(＝O)2-；
R is selected from hydrogen or C1～C12An alkyl group; or R and R3Or R3"looping.
Further: said R1Selected from phenyl, furyl and substituted phenyl; the substituent of the substituted phenyl is selected from halogen and C1～C6Alkyl radical, C1～C6An alkoxy group; said C is1～C6Alkoxy is selected from methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy;
said R2Is selected from C1～C6Alkyl, 5-to 10-membered aryl, substituted 5-to 10-membered aryl, C1～C6The alkyl is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl and hexyl; the substituent for substituting the 5-to 10-membered aryl is selected from C1～C6Alkyl radical, C1～C6Alkoxy, halogen; said C is1～C6Alkoxy is selected from methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy;
said R2' selected from C1～C6Alkyl, 5-to 10-membered aryl, substituted 5-to 10-membered aryl, C1～C6The alkyl is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl and hexyl; the substituent for substituting the 5-to 10-membered aryl is selected from C1～C6Alkyl radical, C1～C6Alkoxy, halogen; said C is1～C6Alkoxy is selected from methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy;
said R3Is selected from C1～C6Alkyl radical, C1～C6Alkoxy radical, said C1～C6The alkyl group is specifically selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, and,A hexyl group; said C is1～C6Alkoxy is specifically selected from methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy.
Said R3' selected from C1～C6Alkyl radical, C1～C6Alkoxy radical, said C1～C6The alkyl is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl and hexyl; said C is1～C6Alkoxy is specifically selected from methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy.
More specifically:
the On-DNA aldehyde compound is selected from:
the amine-based compound is selected from:
the active hydrogen compound is selected from:
the On-DNA Mannich reaction method provided by the invention comprises the following steps: adding 10-1000 times molar equivalent of amino compound and 0-100 times molar equivalent of catalyst into an On-DNA aldehyde compound solution with molar equivalent of 1 and molar concentration of 0.5-5mM for reaction for 0.5-2 hours, then adding 10-1000 times molar equivalent of active hydrogen compound, and reacting for 1-24 hours at 5-100 ℃.
Preferably, the reaction is carried out in a solvent, wherein the solvent is an aqueous mixed solvent of any one or more of water, methanol, ethanol, acetonitrile, acetone, N-dimethylacetamide, dimethyl sulfoxide, NMP, THF, an inorganic salt buffer solution, an organic acid buffer solution and an organic base buffer solution. Preferably, the solvent comprises PBS buffer, BBS buffer or MES solution.
Preferably, the catalyst is selected from p-toluenesulfonic acid, L-proline or D-proline.
Preferably, the molar equivalent of the On-DNA aldehyde compound is 1, and the molar equivalent of the amine-based compound is 20 equivalents, 50 equivalents, 100 equivalents, 200 equivalents, 300 equivalents, 400 equivalents, 500 equivalents, 600 equivalents, 800 equivalents; the molar equivalent of the catalyst is 0 equivalent, 1 equivalent, 2 equivalents, 5 equivalents, 10 equivalents, 20 equivalents and 50 equivalents; the molar equivalent of the active hydrogen compound is 20 equivalents, 50 equivalents, 100 equivalents, 200 equivalents, 300 equivalents, 400 equivalents, 500 equivalents, 600 equivalents, or 800 equivalents.
Preferably, the reaction time of 1 to 24 hours is 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 18 hours or 20 hours.
Preferably, the reaction time of 0.5 to 2 hours is 0.6 hour, 0.8 hour, 1.0 hour, 1.2 hour, 1.4 hour, 1.6 hour, 1.8 hour.
Preferably, the reaction temperature of the reaction is 10 ℃,20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃ and 90 ℃.
Further, the above methods are used for single well (single tube) or batch multi-well plate operations.
Further, the above method is used for the synthesis of DNA-encoding compounds or compound libraries for multiwell plates.
The method can realize the construction of the beta-aminocarbonyl compound by the water-phase multi-component Mannich reaction in the construction of the DNA coding compound library, and can introduce aldehyde, amine and a compound containing active hydrogen as a synthesis module in a large scale. The method is efficient, has a single product, can be carried out in a mixed aqueous phase of an organic solvent/aqueous phase, is simple to operate, and is suitable for synthesizing a DNA coding compound library by using a multi-well plate.
Definitions of terms used in connection with the present invention: the initial definitions provided herein for a group or term apply to that group or term throughout the specification unless otherwise indicated; for terms not specifically defined herein, the meanings that would be given to them by a person skilled in the art are to be given in light of the disclosure and the context.
"substituted" means that a hydrogen atom in a molecule is replaced by a different atom or molecule.
The minimum and maximum values of the carbon atom content in the hydrocarbon group are indicated by a prefix, e.g. prefix (Ca-C)b) Alkyl means any alkyl group containing from "a" to "b" carbon atoms. Thus, for example, C1～C12The alkyl group is a straight-chain or branched alkyl group having 1 to 12 carbon atoms.
Alkyl means a straight or branched hydrocarbon radical in an alkane molecule, e.g. methyl-CH3ethyl-CH2CH3methylene-CH2-; the alkyl group may also be part of another group, such as C1～C6Alkoxy radical, C1～C6An alkylamino group.
Cycloalkyl groups: refers to saturated or partially saturated cyclic groups having multiple carbon atoms and no ring heteroatoms, and having a single ring or multiple rings (including fused, bridged, and spiro ring systems).
The halogen is fluorine, chlorine, bromine or iodine.
Alkoxy groups: means that the alkyl radical is bound to an oxygen atom to form a substituent, e.g. methoxy is-OCH3。
Halogenated phenyl group: refers to a group formed by substituting H on a phenyl group with a halogen.
Alkyl phenyl: refers to a group formed by substituting H on a phenyl group with an alkyl group.
Aryl: refers to an aromatic single cyclic or multiple cyclic group composed of C atoms and containing no hetero atom.
The aromatic heterocyclic group is a single cyclic group or a plurality of cyclic groups having aromaticity composed of atoms such as C, O, S, N.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Description of the drawings:
FIG. 1 is the LC-Ms and Ms spectra of Compound 2 of example 1.
The specific implementation mode is as follows:
the technical solution of the present invention is fully and clearly described below with reference to specific examples. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.
DNA-NH in the present invention2Or
Is formed by single-stranded or double-stranded DNA and a linker group and has-NH2DNA constructs for linkers, e.g. DNA-NH of "compound 1" in WO20050584792And (5) structure. Also for example the following DNA structure:
wherein A is adenine, T is thymine, C is cytosine, and G is guanine.
In the invention, the term "room temperature" means 20-30 ℃.
HATU: 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate. DIPEA: n, N-diisopropylethylamine; CAS: 7087-68-5. DMSO, DMSO: dimethyl sulfoxide solution; DMA: and (3) dimethylacetamide. MES: 2- (N-morpholino) ethanesulfonic acid. BBS buffer: a borate buffered solution. NMP: n-methyl pyrrolidone. THF: tetrahydrofuran. PBS buffer: phosphate buffered saline solution.
Example 1 Synthesis of Compound 2
Carboxylic acid (10. mu.L, 100 equivalents, 200mM DMA solution), HATU (5. mu.L, 100 equivalents, 400mM DMA solution) and DIPEA (5. mu.L, 100 equivalents, 400mM DMA solution) were mixed in advance and then stored at-20 ℃ for 5 minutes; reacting the compound DNA-NH2(10. mu.L) was dissolved in borate buffer (10. mu.L, pH 9.4,250mM) to give a starting concentration of 1 mM; adding the mixture to DNA-NH2And (3) in the buffer solution, fully and uniformly mixing the reaction solution, and reacting for 12 hours at room temperature.
After the reaction is finished: precipitating with ethanol, adding 4 μ L of 5M sodium chloride solution, adding 120 μ L of anhydrous ethanol, shaking, freezing at-20 deg.C for 2 hr, centrifuging at high speed for half an hour, and removing supernatant to obtain compound1 with theoretical molecular weight of 5316.0 and actual measured molecular weight of 5315.9.
Compound1 (20. mu.L, 1 equivalent, 1mM MES solution) and p-anisidine (10. mu.L, 100 equivalents, 200mM DMSO solution) were mixed well in advance, and the reaction was left to activate at room temperature for 1 hour. After completion of activation, 1-methoxy-2-propanone (10. mu.L, 100 equivalents, 200mM DMSO solution) was added thereto, and the reaction mixture was mixed well, followed by allowing the reaction mixture to react at room temperature for 16 hours.
After the reaction is finished: precipitating with ethanol, adding 4 μ L of 5M sodium chloride solution into the solution, then continuously adding 120 μ L of anhydrous ethanol, oscillating uniformly, freezing the reaction at-20 deg.C for 2 hr, centrifuging at high speed for half an hour, pouring off the supernatant, and dissolving the rest precipitate with deionized water to obtain compound 2 with a conversion rate of 63%. The theoretical molecular weight is 5509.0, and the measured molecular weight is 5508.7.
Example 2 Synthesis of Compound 2
Compound1 (20. mu.L, 1 equivalent, 1mM in PBS), p-anisidine (10. mu.L, 100 equivalents, 200mM in DMSO) and L-proline (1. mu.L, 5 equivalents, 100mM in DMSO) were mixed well in advance, the reaction mixture was activated at room temperature for 1 hour, after activation, 1-methoxy-2-propanone (10. mu.L, 200 equivalents, 400mM in DMSO) was added thereto, the reaction mixture was mixed well, and the reaction mixture was allowed to react at room temperature for 16 hours.
After the reaction is finished: precipitating with ethanol, adding 4 μ L of 5M sodium chloride solution into the solution, then continuously adding 120 μ L of anhydrous ethanol, oscillating uniformly, freezing the reaction at-20 deg.C for 2 hr, centrifuging at high speed for half an hour, pouring off the supernatant, and dissolving the rest precipitate with deionized water to obtain compound 2 with a conversion rate of 58%. The theoretical molecular weight is 5509.0, and the measured molecular weight is 5508.9.
Example 3 Synthesis of Compound 2
Compound1 (20. mu.L, 1 equivalent, 1mM in PBS), p-anisidine (10. mu.L, 100 equivalents, 200mM in DMSO) and p-toluenesulphonic acid (1. mu.L, 5 equivalents, 100mM in DMSO) were mixed well in advance, and the reaction was left to activate at room temperature for 1 hour. After completion of activation, 1-methoxy-2-propanone (10. mu.L, 200 equivalents, 400mM DMSO solution) was added thereto, and the reaction mixture was thoroughly mixed, followed by allowing the reaction mixture to react at room temperature for 16 hours.
After the reaction is finished: precipitating with ethanol, adding 4 μ L of 5M sodium chloride solution into the solution, then continuously adding 120 μ L of anhydrous ethanol, oscillating uniformly, freezing the reaction at-20 deg.C for 2 hr, centrifuging at high speed for half an hour, pouring off the supernatant, and dissolving the rest precipitate with deionized water to obtain compound 2 with a conversion rate of 58%. The theoretical molecular weight is 5509.0, and the measured molecular weight is 5508.9.
Example 4 Synthesis of Compound 2
Compound1 (20. mu.L, 1 equivalent, 1mM MES solution) and p-anisidine (10. mu.L, 100 equivalents, 200mM DMSO solution) were mixed well in advance, and the reaction mixture was left to activate at room temperature for 1 hour. After completion of activation, 1-methoxy-2-propanone (10. mu.L, 200 equivalents, 400mM DMSO solution) was added thereto, and the reaction mixture was thoroughly mixed, followed by allowing the reaction mixture to react at room temperature for 16 hours.
After the reaction is finished: precipitating with ethanol, adding 4 μ L of 5M sodium chloride solution into the solution, then continuously adding 120 μ L of anhydrous ethanol, oscillating uniformly, freezing the reaction at-20 deg.C for 2 hr, centrifuging at high speed for half an hour, pouring out the supernatant, and dissolving the rest precipitate with deionized water to obtain compound 2 with a conversion rate of 65%. The theoretical molecular weight is 5509.0, and the measured molecular weight is 5508.9.
Example 5 Synthesis of Compound 2
Compound1 (20. mu.L, 1 equivalent, 1mM MES solution) and p-anisidine (10. mu.L, 50 equivalents, 100mM DMSO solution) were mixed well in advance, and the reaction mixture was left to activate at room temperature for 1 hour. After completion of activation, 1-methoxy-2-propanone (10. mu.L, 50 equivalents, 100mM DMSO solution) was added thereto, and the reaction mixture was mixed well, followed by allowing the reaction mixture to react at room temperature for 16 hours.
After the reaction is finished: precipitating with ethanol, adding 4 μ L of 5M sodium chloride solution into the solution, adding 120 μ L of anhydrous ethanol, shaking, freezing at-20 deg.C for 2 hr, centrifuging at high speed for half an hour, removing supernatant, dissolving the precipitate with deionized water to obtain compound 2 with conversion rate of 71%. The theoretical molecular weight is 5509.0, and the measured molecular weight is 5509.1.
Example 6 Synthesis of Compound 2
Compound1 (20. mu.L, 1 equivalent, 1mM MES solution) and p-anisidine (10. mu.L, 200 equivalents, 400mM DMSO solution) were mixed well in advance, and the reaction mixture was left to activate at room temperature for 1 hour. After completion of activation, 1-methoxy-2-propanone (10. mu.L, 200 equivalents, 400mM DMSO solution) was added thereto, and the reaction mixture was thoroughly mixed, followed by allowing the reaction mixture to react at room temperature for 16 hours.
After the reaction is finished: precipitating with ethanol, adding 4 μ L of 5M sodium chloride solution into the solution, adding 120 μ L of anhydrous ethanol, shaking, freezing at-20 deg.C for 2 hr, centrifuging at high speed for half an hour, removing supernatant, dissolving the precipitate with deionized water to obtain compound 2 with conversion rate of 95%. The theoretical molecular weight is 5509.0, and the measured molecular weight is 5509.2.
Example 7 Synthesis of Compound 2
Compound1 (20. mu.L, 1 equivalent, 1mM MES solution) and p-anisidine (10. mu.L, 400 equivalent, 800mM DMSO solution) were mixed well in advance, the reaction was left to activate at room temperature for 1 hour, after activation, 1-methoxy-2-propanone (10. mu.L, 400 equivalent, 800mM DMSO solution) was added thereto, the reaction was mixed well, and then the reaction was left to react at room temperature for 16 hours.
After the reaction is finished: precipitating with ethanol, adding 4 μ L of 5M sodium chloride solution into the solution, then continuously adding 120 μ L of anhydrous ethanol, oscillating uniformly, freezing the reaction at-20 deg.C for 2 hr, centrifuging at high speed for half an hour, pouring off the supernatant, and dissolving the rest precipitate with deionized water to obtain compound 2 with a conversion rate of 94%. The theoretical molecular weight is 5509.0, and the measured molecular weight is 5509.1.
Example 8 Synthesis of Compound 2
Compound1 (20. mu.L, 1 equivalent, 1mM MES solution) and p-anisidine (10. mu.L, 100 equivalents, 200mM DMSO solution) were mixed well in advance, and the reaction mixture was left to activate at room temperature for 1 hour. After completion of activation, 1-methoxy-2-propanone (10. mu.L, 100 equivalents, 200mM DMSO solution) was added thereto, and the reaction mixture was mixed well, followed by reaction at 40 ℃ for 16 hours.
After the reaction is finished: precipitating with ethanol, adding 4 μ L of 5M sodium chloride solution into the solution, then continuously adding 120 μ L of anhydrous ethanol, oscillating uniformly, freezing the reaction at-20 deg.C for 2 hr, centrifuging at high speed for half an hour, pouring out the supernatant, and dissolving the rest precipitate with deionized water to obtain compound 2 with a conversion rate of 88%. The theoretical molecular weight is 5509.0, and the measured molecular weight is 5509.1.
Example 9 Synthesis of Compound 4
Carboxylic acid (10. mu.L, 100 equivalents, 200mM DMA solution), HATU (5. mu.L, 100 equivalents, 400mM DMA solution) and DIPEA (5. mu.L, 100 equivalents, 400mM DMA solution) were mixed in advance and then stored at-20 ℃ for 5 minutes; reacting the compound DNA-NH2(10. mu.L) was dissolved in borate buffer (10. mu.L, pH 9.4,250mM) to give a starting concentration of 1 mM; adding the mixture to DNA-NH2And (3) in the buffer solution, fully and uniformly mixing the reaction solution, and reacting for 12 hours at room temperature.
After the reaction is finished: precipitating with ethanol, adding 4 μ L of 5M sodium chloride solution, adding 120 μ L of anhydrous ethanol, shaking, freezing at-20 deg.C for 2 hr, centrifuging at high speed for half an hour, and removing supernatant to obtain compound 3 with theoretical molecular weight of 5334.0 and actual measured molecular weight of 5334.7.
Compound 3 was dissolved in deionized water (10. mu.L) to prepare an initial concentration of 2mM, Compound 3 (20. mu.L, 1 equivalent, 1mM MES solution) was mixed with p-anisidine (10. mu.L, 100 equivalents, 200mM DMSO solution) in advance, and the reaction mixture was allowed to stand at room temperature for 1 hour. After completion of activation, 1-methoxy-2-propanone (10. mu.L, 100 equivalents, 200mM DMSO solution) was added thereto, and the reaction mixture was mixed well, followed by allowing the reaction mixture to react at room temperature for 16 hours.
After the reaction is finished: precipitating with ethanol, adding 4 μ L of 5M sodium chloride solution into the solution, then continuously adding 120 μ L of anhydrous ethanol, oscillating uniformly, freezing the reaction at-20 deg.C for 2 hr, centrifuging at high speed for half an hour, pouring off the supernatant, and dissolving the rest precipitate with deionized water to obtain compound 4 with conversion rate of 66%. The theoretical molecular weight is 5527.0, and the measured molecular weight is 5526.7.
Example 10 Synthesis of Compound 6
Carboxylic acid (10. mu.L, 100 equivalents, 200mM DMA solution), HATU (5. mu.L, 100 equivalents, 400mM DMA solution) and DIPEA (5. mu.L, 100 equivalents, 400mM DMA solution) were mixed in advance and then stored at-20 ℃ for 5 minutes; reacting the compound DNA-NH2(10. mu.L) was dissolved in borate buffer (10. mu.L, pH 9.4,250mM) to give a starting concentration of 1 mM; adding the mixture to DNA-NH2And (3) in the buffer solution, fully and uniformly mixing the reaction solution, and reacting for 12 hours at room temperature.
After the reaction is finished: precipitating with ethanol, adding 4 μ L of 5M sodium chloride solution, adding 120 μ L of anhydrous ethanol, shaking, freezing at-20 deg.C for 2 hr, centrifuging at high speed for half an hour, and removing supernatant to obtain compound 5 with theoretical molecular weight of 5306.0 and actual measured molecular weight of 5306.8.
Compound 5 was dissolved in deionized water (10. mu.L) to make a starting concentration of 2 mM. Compound 5 (20. mu.L, 1 equivalent, 1mM MES solution) was mixed with p-anisidine (10. mu.L, 100 equivalents, 200mM DMSO solution) in advance, and the reaction mixture was left to activate at room temperature for 1 hour. After completion of activation, 1-methoxy-2-propanone (10. mu.L, 100 equivalents, 200mM DMSO solution) was added thereto, and the reaction mixture was mixed well, followed by allowing the reaction mixture to react at room temperature for 16 hours.
After the reaction is finished: precipitating with ethanol, adding 4 μ L of 5M sodium chloride solution into the solution, then continuously adding 120 μ L of anhydrous ethanol, oscillating uniformly, freezing the reaction at-20 deg.C for 2 hr, centrifuging at high speed for half an hour, pouring off the supernatant, and dissolving the rest precipitate with deionized water to obtain compound 6 with a conversion rate of 52%. The theoretical molecular weight is 5499.0, and the measured molecular weight is 5499.3.
Example 11 Synthesis of Compound 8
Carboxylic acid (10. mu.L, 100 equivalents, 200mM DMA solution), HATU (5. mu.L, 100 equivalents, 400mM DMA solution) and DIPEA (5. mu.L, 100 equivalents, 400mM DMA solution) were mixed in advance and then stored at-20 ℃ for 5 minutes; reacting the compound DNA-NH2(10. mu.L) was dissolved in borate buffer (10. mu.L, pH 9.4,250mM) to give a starting concentration of 1 mM; adding the mixture to DNA-NH2And (3) in the buffer solution, fully and uniformly mixing the reaction solution, and reacting for 12 hours at room temperature.
After the reaction is finished: precipitating with ethanol, adding 4 μ L of 5M sodium chloride solution, adding 120 μ L of anhydrous ethanol, shaking, freezing at-20 deg.C for 2 hr, centrifuging at high speed for half an hour, and removing supernatant to obtain compound 7 with theoretical molecular weight of 5334.0 and actual measured molecular weight of 5334.2.
Compound 7 was dissolved in deionized water (10. mu.L) to prepare an initial concentration of 2mM, Compound 7 (20. mu.L, 1 equivalent, 1mM MES solution) was mixed with p-propylaniline (10. mu.L, 100 equivalents, 200mM DMSO solution) to homogeneity, and the reaction was allowed to stand at room temperature for 1 hour. After completion of activation, 1-methoxy-2-propanone (10. mu.L, 100 equivalents, 200mM DMSO solution) was added thereto, and the reaction mixture was mixed well, followed by allowing the reaction mixture to react at room temperature for 16 hours.
After the reaction is finished: precipitating with ethanol, adding 4 μ L of 5M sodium chloride solution into the solution, adding 120 μ L of anhydrous ethanol, oscillating, freezing the reaction at-20 deg.C for 2 hr, centrifuging at high speed for half an hour, removing supernatant, and dissolving the precipitate with deionized water to obtain compound 8 with conversion rate of 80%. The theoretical molecular weight is 5539.0, and the measured molecular weight is 5538.9.
Example 12 Synthesis of Compound 10
Carboxylic acid (10. mu.L, 100 equivalents, 200mM DMA solution), HATU (5. mu.L, 100 equivalents, 400mM DMA solution) and DIPEA (5. mu.L, 100 equivalents, 400mM DMA solution) were mixed in advance and then stored at-20 ℃ for 5 minutes; reacting the compound DNA-NH2(10. mu.L) was dissolved in borate buffer (10. mu.L, pH 9.4,250mM) to give a starting concentration of 1 mM; adding the mixture to DNA-NH2And (3) in the buffer solution, fully and uniformly mixing the reaction solution, and reacting for 12 hours at room temperature.
After the reaction is finished: precipitating with ethanol, adding 4 μ L of 5M sodium chloride solution, adding 120 μ L of anhydrous ethanol, shaking, freezing at-20 deg.C for 2 hr, centrifuging at high speed for half an hour, and removing supernatant to obtain compound 9 with theoretical molecular weight of 5346.0 and actual measured molecular weight of 5345.8.
Compound 9 was dissolved in deionized water (10. mu.L) to prepare an initial concentration of 2mM, Compound 9 (20. mu.L, 1 equivalent, 1mM MES solution) was mixed with p-propylaniline (10. mu.L, 100 equivalents, 200mM DMSO solution) to homogeneity, and the reaction was allowed to stand at room temperature for 1 hour. After completion of activation, 1-methoxy-2-propanone (10. mu.L, 100 equivalents, 200mM DMSO solution) was added thereto, and the reaction mixture was mixed well, followed by allowing the reaction mixture to react at room temperature for 16 hours.
After the reaction is finished: precipitating with ethanol, adding 4 μ L of 5M sodium chloride solution into the solution, then continuously adding 120 μ L of anhydrous ethanol, oscillating uniformly, freezing the reaction at-20 deg.C for 2 hr, centrifuging at high speed for half an hour, pouring off the supernatant, and dissolving the rest precipitate with deionized water to obtain compound 10 with a conversion rate of 53%. The theoretical molecular weight is 5551.0, and the measured molecular weight is 5550.6.
Example 13 Synthesis of Compound 12
Carboxylic acid (10. mu.L, 100 equivalents, 200mM DMA solution), HATU (5. mu.L, 100 equivalents, 400mM DMA solution) and DIPEA (5. mu.L, 100 equivalents, 400mM DMA solution) were mixed in advance and then stored at-20 ℃ for 5 minutes; reacting the compound DNA-NH2(10. mu.L) was dissolved in borate buffer (10. mu.L, pH 9.4,250mM) to give a starting concentration of 1 mM; adding the mixture to DNA-NH2And (3) in the buffer solution, fully and uniformly mixing the reaction solution, and reacting for 12 hours at room temperature.
After the reaction is finished: precipitating with ethanol, adding 4 μ L of 5M sodium chloride solution, adding 120 μ L of anhydrous ethanol, shaking, freezing at-20 deg.C for 2 hr, centrifuging at high speed for half an hour, and removing supernatant to obtain compound 11 with theoretical molecular weight of 5346.0 and actual measured molecular weight of 5346.3.
Compound 11 was dissolved in deionized water (10. mu.L) to prepare an initial concentration of 2mM, Compound 11 (20. mu.L, 1 equivalent, 1mM MES solution) was mixed with p-propylaniline (10. mu.L, 100 equivalents, 200mM DMSO solution) to homogeneity, and the reaction was allowed to stand at room temperature for 1 hour. After completion of activation, 1-methoxy-2-propanone (10. mu.L, 100 equivalents, 200mM DMSO solution) was added thereto, and the reaction mixture was mixed well, followed by allowing the reaction mixture to react at room temperature for 16 hours.
After the reaction is finished: precipitating with ethanol, adding 4 μ L of 5M sodium chloride solution into the solution, adding 120 μ L of anhydrous ethanol, oscillating, freezing at-20 deg.C for 2 hr, centrifuging at high speed for half an hour, removing supernatant, and dissolving the precipitate with deionized water to obtain compound 12 with conversion rate of 44%. The theoretical molecular weight is 5551.0, and the measured molecular weight is 5551.0.
Example 14 Synthesis of Compound 13
Compound 3 (20. mu.L, 1 equivalent, 1mM MES solution) was mixed with p-propylaniline (10. mu.L, 100 equivalents, 200mM DMSO solution) in advance, and the reaction mixture was left to stand at room temperature for 1 hour. After completion of activation, 1-methoxy-2-propanone (10. mu.L, 100 equivalents, 200mM DMSO solution) was added thereto, and the reaction mixture was mixed well, followed by allowing the reaction mixture to react at room temperature for 16 hours.
After the reaction is finished: precipitating with ethanol, adding 4 μ L of 5M sodium chloride solution into the solution, then continuously adding 120 μ L of anhydrous ethanol, oscillating uniformly, freezing the reaction at-20 deg.C for 2 hr, centrifuging at high speed for half an hour, pouring off the supernatant, and dissolving the rest precipitate with deionized water to obtain compound 13 with a conversion rate of 90%. The theoretical molecular weight is 5539.0, and the measured molecular weight is 5538.7.
Example 15 Synthesis of Compound 14
Compound 3 (20. mu.L, 1 equivalent, 1mM MES solution) was mixed well with 5-aminoindand (10. mu.L, 100 equivalents, 200mM DMSO solution), and the reaction was left to activate at room temperature for 1 hour. After completion of activation, 1-methoxy-2-propanone (10. mu.L, 100 equivalents, 200mM DMSO solution) was added thereto, and the reaction mixture was mixed well, followed by allowing the reaction mixture to react at room temperature for 16 hours.
After the reaction is finished: precipitating with ethanol, adding 4 μ L of 5M sodium chloride solution into the solution, adding 120 μ L of anhydrous ethanol, shaking, freezing at-20 deg.C for 2 hr, centrifuging at high speed for half an hour, removing supernatant, and dissolving the precipitate with deionized water to obtain compound 14 with conversion rate of 74%. The theoretical molecular weight is 5537.0, and the measured molecular weight is 5536.7.
Example 16 Synthesis of Compound 15
Compound 3 (20. mu.L, 1 equivalent, 1mM MES solution) and 4-fluoroaniline (10. mu.L, 100 equivalents, 200mM DMSO solution) were mixed well in advance, and the reaction mixture was left to activate at room temperature for 1 hour. After completion of activation, 1-methoxy-2-propanone (10. mu.L, 100 equivalents, 200mM DMSO solution) was added thereto, and the reaction mixture was mixed well, followed by allowing the reaction mixture to react at room temperature for 16 hours.
After the reaction is finished: precipitating with ethanol, adding 4 μ L of 5M sodium chloride solution into the solution, then continuously adding 120 μ L of anhydrous ethanol, oscillating uniformly, freezing the reaction at-20 deg.C for 2 hr, centrifuging at high speed for half an hour, pouring off the supernatant, and dissolving the rest precipitate with deionized water to obtain compound 15 with a conversion rate of 36%. The theoretical molecular weight is 5515.0, and the measured molecular weight is 5514.7.
Example 17 Synthesis of Compound 16
Compound1 (20. mu.L, 1 equivalent, 1mM MES solution) and aniline (10. mu.L, 100 equivalents, 200mM DMSO solution) were mixed well in advance, and the reaction was left to activate at room temperature for 1 hour. After completion of activation, 1-methoxy-2-propanone (10. mu.L, 100 equivalents, 200mM DMSO solution) was added thereto, and the reaction mixture was mixed well, followed by allowing the reaction mixture to react at room temperature for 16 hours.
After the reaction is finished: precipitating with ethanol, adding 4 μ L of 5M sodium chloride solution into the solution, adding 120 μ L of anhydrous ethanol, oscillating, freezing the reaction at-20 deg.C for 2 hr, centrifuging at high speed for half an hour, removing supernatant, and dissolving the precipitate with deionized water to obtain compound 16 with conversion rate of 59%. The theoretical molecular weight is 5479.0, and the measured molecular weight is 5478.7.
Example 18 Synthesis of Compound 17
Compound 7 (20. mu.L, 1 equivalent, 1mM MES solution) was mixed well with 5-aminoindand (10. mu.L, 100 equivalents, 200mM DMSO solution), and the reaction was left to activate at room temperature for 1 hour. After completion of activation, 1-methoxy-2-propanone (10. mu.L, 100 equivalents, 200mM DMSO solution) was added thereto, and the reaction mixture was mixed well, followed by allowing the reaction mixture to react at room temperature for 16 hours.
After the reaction is finished: precipitating with ethanol, adding 4 μ L of 5M sodium chloride solution into the solution, then continuously adding 120 μ L of anhydrous ethanol, oscillating uniformly, freezing the reaction at-20 deg.C for 2 hours, centrifuging at high speed for half an hour, pouring out the supernatant, and dissolving the rest precipitate with deionized water to obtain compound 17 with a conversion rate of 98%. The theoretical molecular weight is 5537.0, and the measured molecular weight is 5537.2.
Example 19 Synthesis of Compound 18
Compound 7 (20. mu.L, 1 equivalent, 1mM MES solution) and 4-fluoroaniline (10. mu.L, 100 equivalents, 200mM DMSO solution) were mixed well in advance, and the reaction mixture was left to activate at room temperature for 1 hour. After completion of activation, 1-methoxy-2-propanone (10. mu.L, 100 equivalents, 200mM DMSO solution) was added thereto, and the reaction mixture was mixed well, followed by allowing the reaction mixture to react at room temperature for 16 hours.
After the reaction is finished: precipitating with ethanol, adding 4 μ L of 5M sodium chloride solution into the solution, adding 120 μ L of anhydrous ethanol, shaking, freezing at-20 deg.C for 2 hr, centrifuging at high speed for half an hour, removing supernatant, and dissolving the precipitate with deionized water to obtain compound 18 with conversion rate of 29%. The theoretical molecular weight is 5515.0, and the measured molecular weight is 5515.1.
Example 20 Synthesis of Compound 19
Compound 9 (20. mu.L, 1 equivalent, 1mM MES solution) was mixed well with 5-aminoindand (10. mu.L, 100 equivalents, 200mM DMSO solution), and the reaction was left to activate at room temperature for 1 hour. After completion of activation, 1-methoxy-2-propanone (10. mu.L, 100 equivalents, 200mM DMSO solution) was added thereto, and the reaction mixture was mixed well, followed by allowing the reaction mixture to react at room temperature for 16 hours.
After the reaction is finished: precipitating with ethanol, adding 4 μ L of 5M sodium chloride solution into the solution, adding 120 μ L of anhydrous ethanol, shaking, freezing at-20 deg.C for 2 hr, centrifuging at high speed for half an hour, removing supernatant, and dissolving the precipitate with deionized water to obtain compound 19 with conversion rate of 51%. The theoretical molecular weight is 5549.0, and the measured molecular weight is 5548.9.
Example 21 Synthesis of Compound 20
Compound 9 (20. mu.L, 1 equivalent, 1mM MES solution) and 4-fluoroaniline (10. mu.L, 100 equivalents, 200mM DMSO solution) were mixed well in advance, and the reaction mixture was left to activate at room temperature for 1 hour. After completion of activation, 1-methoxy-2-propanone (10. mu.L, 100 equivalents, 200mM DMSO solution) was added thereto, and the reaction mixture was mixed well, followed by allowing the reaction mixture to react at room temperature for 16 hours.
After the reaction is finished: precipitating with ethanol, adding 4 μ L of 5M sodium chloride solution into the solution, adding 120 μ L of anhydrous ethanol, oscillating, freezing the reaction at-20 deg.C for 2 hr, centrifuging at high speed for half an hour, removing supernatant, and dissolving the precipitate with deionized water to obtain compound 20 with conversion rate of 43%. The theoretical molecular weight is 5527.0, and the measured molecular weight is 5527.1.
Example 22 Synthesis of Compound 21
Compound 11 (20. mu.L, 1 equivalent, 1mM MES solution) was mixed well with 5-aminoindand (10. mu.L, 100 equivalents, 200mM DMSO solution), and the reaction was left to activate at room temperature for 1 hour. After completion of activation, 1-methoxy-2-propanone (10. mu.L, 100 equivalents, 200mM DMSO solution) was added thereto, and the reaction mixture was mixed well, followed by allowing the reaction mixture to react at room temperature for 16 hours.
After the reaction is finished: precipitating with ethanol, adding 4 μ L of 5M sodium chloride solution into the solution, adding 120 μ L of anhydrous ethanol, oscillating, freezing at-20 deg.C for 2 hr, centrifuging at high speed for half an hour, removing supernatant, and dissolving the precipitate with deionized water to obtain compound 21 with conversion rate of 44%. The theoretical molecular weight is 5549.0, and the measured molecular weight is 5549.0.
Example 23 Synthesis of Compound 22
Compound 11 (20. mu.L, 1 equivalent, 1mM MES solution) and 4-fluoroaniline (10. mu.L, 100 equivalents, 200mM DMSO solution) were mixed well in advance, and the reaction mixture was left to activate at room temperature for 1 hour. After completion of activation, 1-methoxy-2-propanone (10. mu.L, 100 equivalents, 200mM DMSO solution) was added thereto, and the reaction mixture was mixed well, followed by allowing the reaction mixture to react at room temperature for 16 hours.
After the reaction is finished: precipitating with ethanol, adding 4 μ L of 5M sodium chloride solution into the solution, adding 120 μ L of anhydrous ethanol, shaking, freezing at-20 deg.C for 2 hr, centrifuging at high speed for half an hour, removing supernatant, and dissolving the precipitate with deionized water to obtain compound 22 with conversion rate of 28%. The theoretical molecular weight is 5527.0, and the measured molecular weight is 5527.1.
Example 24 Synthesis of Compound 23
Compound1 (20. mu.L, 1 equivalent, 1mM MES solution) and p-anisidine (10. mu.L, 100 equivalents, 200mM DMSO solution) were mixed well in advance, and the reaction was left to activate at room temperature for 1 hour. After completion of activation, cyclohexanone (10. mu.L, 100 equivalents, 200mM DMSO solution) was added thereto, and the reaction solution was mixed well, followed by allowing the reaction solution to react at room temperature for 16 hours.
After the reaction is finished: precipitating with ethanol, adding 4 μ L of 5M sodium chloride solution into the solution, then continuously adding 120 μ L of anhydrous ethanol, oscillating uniformly, freezing the reaction at-20 deg.C for 2 hr, centrifuging at high speed for half an hour, pouring off the supernatant, and dissolving the rest precipitate with deionized water to obtain compound 23 with a conversion rate of 59%. The theoretical molecular weight is 5509.0, and the measured molecular weight is 5509.0.
Claims (10)
1. A method of On-DNA Mannich reaction, characterized in that: the reaction takes an On-DNA aldehyde compound as a raw material, and the On-DNA aldehyde compound reacts with an active hydrogen compound and an amino compound to obtain an On-DNA product; the structural formula of the On-DNA aldehyde compound is DNA-R1-CHO, the structural formula of the amino compound is R2R2 ’NH, the structural formula of the active hydrogen compound is
The structural formula of the On-DNA product is shown in the specification
Wherein the DNA in the structural formula comprises a single-stranded or double-stranded nucleotide chain obtained by polymerizing artificially modified and/or unmodified nucleotide monomers, and the nucleotide chain is connected with R through one or more chemical bonds or groups1Connecting;
said R1Is a group with the molecular weight below 1000 directly connected with DNA and aldehyde group carbon atom or no group;
said R2、R2' is hydrogen or a group having a molecular weight of 1000 or less directly bonded to the nitrogen atom of the amino group;
said R3、R3' is hydrogen or a group having a molecular weight of 1000 or less directly bonded to the carbon atom on which the active hydrogen is present;
the EWG is an electron-withdrawing group with the molecular weight of below 1000 and directly connected with a carbon atom.
2. The method of claim 1, wherein: said R1、R2、R2＇、R3、R3Each independently selected from an alkyl group, a substituted alkyl group, a 5-to 10-membered aryl group, a substituted 5-to 10-membered aryl group, a 5-to 10-membered aromatic heterocyclic group, and a substituted 5-to 10-membered aromatic heterocyclic group; wherein the alkyl is C1～C20Alkyl or C3～C8A cycloalkyl group; the number of substituents of the substituted alkyl group is one or more; the substituent of the substituted alkyl is one or more independently selected from halogen, carboxyl, nitro, alkoxy, halogenated phenyl, phenyl and alkyl phenyl; the number of the substituent for substituting the 5-to 10-membered aryl is one or more, and the substituents for substituting the 5-to 10-membered aryl are independently selected from halogen, cyano, nitro, carboxyl, alkoxy and C1～C20One or more of alkyl and trifluoromethyl; the number of the substituent(s) for substituting the 5-to 10-membered aromatic heterocyclic group is one or more, and the substituent(s) for substituting the 5-to 10-membered aromatic heterocyclic group are independently selected from the group consisting of halogen, cyano, nitro, carboxyl, alkoxy, C1～C20One or more of alkyl and trifluoromethyl;
said EWG is-NO2、-C(O)R、-CN、-C(O)OR、R-S(＝O)2-；
R is selected from hydrogen or C1～C12An alkyl group; or R and R3Or R3"looping.
3. The method of claim 2, wherein: said R1Selected from phenyl, furyl and substituted phenyl; the substituent of the substituted phenyl is selected from halogen and C1～C6Alkyl radical, C1～C6An alkoxy group; said C is1～C6Alkoxy is selected from methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy;
said R2Is selected from C1～C6Alkyl, 5-to 10-membered aryl, substituted 5-to 10-membered aryl, C1～C6The alkyl is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl and hexyl; the substituent for substituting the 5-to 10-membered aryl is selected from C1～C6Alkyl radical, C1～C6Alkoxy, halogen; said C is1～C6Alkoxy is selected from methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy;
said R2' selected from C1～C6Alkyl, 5-to 10-membered aryl, substituted 5-to 10-membered aryl, C1～C6The alkyl is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl and hexyl; the substituent for substituting the 5-to 10-membered aryl is selected from C1～C6Alkyl radical, C1～C6Alkoxy, halogen; said C is1～C6Alkoxy is selected from methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy;
said R3Is selected from C1～C6Alkyl radical, C1～C6Alkoxy radical, said C1～C6The alkyl is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl and hexyl; said C is1～C6Alkoxy is selected from methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy;
said R3' selected from C1～C6Alkyl radical, C1～C6Alkoxy radical, said C1～C6The alkyl is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl and hexyl; said C is1～C6Alkoxy is specifically selected from methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy.
4. The method of claim 1, wherein: the reaction comprises the following reaction steps: adding 10-1000 times molar equivalent of amino compound and 0-100 times molar equivalent of catalyst into an On-DNA aldehyde compound solution with molar equivalent of 1 and molar concentration of 0.5-5mM for reaction for 0.5-2 hours, then adding 10-1000 times molar equivalent of active hydrogen compound, and reacting for 1-24 hours at 5-100 ℃.
5. The method of claim 4, wherein: the catalyst is L-proline, D-proline or p-methyl benzene sulfonic acid.
6. The method of claim 4, wherein: the reaction is carried out in a solvent, and the solvent is one or a mixture of water, methanol, ethanol, acetonitrile, acetone, N-dimethylacetamide, dimethyl sulfoxide, NMP, THF, an inorganic salt buffer solution, an organic acid buffer solution and an organic base buffer solution.
7. The method of claim 4, wherein: the molar equivalent of the On-DNA aldehyde compound is 1, and the molar equivalents of the amine compound are 20 equivalents, 50 equivalents, 100 equivalents, 200 equivalents, 300 equivalents, 400 equivalents, 500 equivalents, 600 equivalents and 800 equivalents; the molar equivalent of the catalyst is 0 equivalent, 1 equivalent, 2 equivalents, 5 equivalents, 10 equivalents, 20 equivalents and 50 equivalents; the molar equivalent of the active hydrogen compound is 20 equivalents, 50 equivalents, 100 equivalents, 200 equivalents, 300 equivalents, 400 equivalents, 500 equivalents, 600 equivalents, or 800 equivalents.
8. The method of claim 4, wherein: the reaction time of 1 to 24 hours is 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 18 hours, or 20 hours.
9. The method according to any one of claims 1-8, wherein: the method may be used in single tube operations or in batch multi-well plate operations.
10. The method according to any one of claims 1-8, wherein: the method is useful for the synthesis of DNA encoding compounds or libraries of compounds.
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